U.S. patent number 5,086,476 [Application Number 07/315,289] was granted by the patent office on 1992-02-04 for method and apparatus for determining a proliferation index of a cell sample.
This patent grant is currently assigned to Cell Analysis Systems, Inc.. Invention is credited to James W. Bacus.
United States Patent |
5,086,476 |
Bacus |
February 4, 1992 |
Method and apparatus for determining a proliferation index of a
cell sample
Abstract
An image processing method and apparatus determines a
proliferation index of a cell sample by staining the cells with a
chromogen for a proliferation substance and a counterstain for the
cell nuclei. The chromogen is activated by an antibody-enzyme
conjugate which binds to the proliferation substance to produce a
stained cell sample. The stained cell sample is examined with an
optical microscope, forming a portion of the apparatus, which
produces a magnified cell sample image. The apparatus optically
filters the cell sample image and produces a pair of optically
enhanced proliferation substance and cell nuclei images. The
enhanced images are electronically analyzed to determine the
amounts of cell nuclei and proliferation substance appearing in the
images, respectively. The amounts are then compared to yield a
proliferation index for the portion of the cell sample appearing in
the cell sample image.
Inventors: |
Bacus; James W. (Hinsdale,
IL) |
Assignee: |
Cell Analysis Systems, Inc.
(Lombard, IL)
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Family
ID: |
27380174 |
Appl.
No.: |
07/315,289 |
Filed: |
February 24, 1989 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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121674 |
Nov 17, 1987 |
5016283 |
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106717 |
Oct 6, 1987 |
5008185 |
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927285 |
Nov 4, 1986 |
5018209 |
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794937 |
Nov 4, 1985 |
4741043 |
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121674 |
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927285 |
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Current U.S.
Class: |
382/133; 356/39;
D14/306 |
Current CPC
Class: |
G01N
15/1468 (20130101); G06K 9/00127 (20130101); G01N
33/56972 (20130101); G01N 33/56966 (20130101); G01N
15/1475 (20130101); G01N 21/6458 (20130101); G01N
2201/127 (20130101) |
Current International
Class: |
G01N
15/14 (20060101); G01N 33/569 (20060101); G06K
9/00 (20060101); G06K 009/00 () |
Field of
Search: |
;382/6,1,8
;364/413.08,413.07,313.09,413.1 ;356/39,40,432,410
;128/665,633,653R,653A ;358/101 ;250/237R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
King and Green, "Monoclonal Antibodies Localize Oestrogen Receptor
in the Nuclei of Target Cells", Nature 307:745-747, 1984. .
Jensen et al., "Receptors Reconsidered: A 20-Year Perspective",
Recent Progress in Hormone Research 38:1-39. .
Greene et al., "Monoclonal Antibodies to Human Estrogen Receptor",
Proc. Natl. Acad. Sci. U.S.A 77:5115-5119, 1980. .
James and Goldstein, "Haemoglobin Content of Individual
Erythrocytes in Normal and Abnormal Blood", British Journal of
Haemotology 28:89-102, 1974. .
McCarty et al., "Estrogen Receptor Analyses", Arch Pathol Lab Med
109:716-721, 1985. .
"Immunocytochemical Assay for the Detection of Human Estrogen
Receptor", Abbott Laboratories, 83-1547/R2, 1986. .
Sherrod and Taylor, "Nonlymphocyte Tumor Markers in Tissues",
Immunopathology and Immunohistology, Chap. 145, pp. 938-947. .
King et al., "Comparison of Immunocytochemical and Steroid-binding
Assays for Estrogen Receptor in Human Breast Tumors", Cancer
Research 45:293-294, 1982. .
Thorell, B., "Cell Studies with Microspectrography", pp.
95-119..
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Primary Examiner: Razavi; Michael
Attorney, Agent or Firm: Fitch, Even, Tabin &
Flannery
Parent Case Text
CROSS REFERENCES TO RELATED APPLICATIONS
This is a continuation-in-part of copending U.S. application Ser.
No. 121,674, filed Nov. 7, 1987 and now U.S. Pat. No. 5.016,283 in
the names of James W. Bacus and Robert J. Marder for Methods and
Apparatus for Immunoploidy Analysis; a continuation-in-part of
copending U.S. application Ser. No. 106,717, filed Oct. 6, 1987 and
now U.S. pat. No. 5,008,185 in the name of James W. Bacus for
Methods and Apparatus for the Quantitation of Nuclear Protein; and
a continuation-in-part of copending U.S. application Ser. No.
927,285, filed Nov. 4, 1986 and now U.S. pat. No. 5,018,209 in the
name of James W. Bacus for Analysis Method and Apparatus for
Biological Specimens.
U.S. application Ser. No. 121,674, filed Nov. 17, 1987, in turn, is
a continuation-in-part of U.S. application Ser. No. 927,285, filed
Nov. 4, 1986 in the name of James W. Bacus for Analysis Method and
Apparatus for Biological Specimens; which is a continuation-in-part
of U.S. application Ser. No. 794,937, filed Nov. 4, 1985 in the
name of James W. Bacus for Method of and an Apparatus for Image
Analyses of Biological Specimens, now U.S. Pat. No. 4,741,043; all
of which are commonly assigned. The disclosures of each of the
aforementioned applications are hereby expressly incorporated
herein by reference.
Claims
What is claimed is:
1. An apparatus for determining a proliferation index of a cell
sample, comprising:
first means for optically sensing portions of a cell sample having
a proliferation optically enhanced substance thereon and producing
a proliferation substance signal corresponding thereto wherein the
first means further comprises an image enhancing optical filter
allowing transmission of light for an optical absorbing region of a
chromogen associated with the optically-enhanced proliferation
substance at a reduced optical absorbing transmission region of a
stain optically marking the cell nuclei;
second means for optically sensing portions of a cell sample having
optically marked cell nuclei and producing a cell nuclei signal
corresponding thereto wherein the second means further comprises an
optical filter allowing transmission of light at an optical
absorbing region of a stain, which is optically marking the cell
nuclei, and at an optical absorbing region of a chromogen
associated with the optically-enhanced proliferation substance;
first determining means coupled to the first sensing means for
determining an amount of the proliferation substance and producing
a proliferation substance amount signal corresponding thereto;
second determining means coupled to the second sensing means for
determining an amount of optically marked cell nuclei and producing
a cell nuclei amount signal corresponding thereto; and
proliferation index determining means coupled to the first
determining means and receiving the proliferation substance amount
signal and coupled to the second determining means and receiving
the cell nuclei amount signal therefrom, for determining a
proliferation index from the proliferation substance amount signal
and the cell nuclei amount signal.
2. An apparatus for determining a proliferation index of a cell
sample as defined in claim 1, wherein the first means further
comprises an image enhancing optical filter which allows
transmission of light at an optical absorbing region of a chromogen
associated with the proliferation substance and at an essentially
one hundred percent optical transmission region of a stain
optically marking the cell nuclei.
3. An apparatus for determining a proliferation index of a cell
sample as defined in claim 2, wherein the first means further
comprises means for storing a digitized image of the
optically-enhanced proliferatino substance.
4. An apparatus for determining a proliferation index of a cell
sample as defined in claim 1, wherein the first determining means
further comprises means for determining an image area occupied by
the optically-enhanced proliferation substance and wherein the
proliferation substance amount signal is indicative of the image
area occupied by the optically-enhanced proliferation
substance.
5. An apparatus for determining a proliferation index of a cell
sample as defined in claim 4, wherein the second determining means
further comprises means for determining an image area occupied by
the optically marked cell nuclei and the cell nuclei amount signal
is indicative of the image area occupied by the optically marked
cell nuclei.
6. An apparatus for determining a proliferation index of a cell
sample as defined in claim 1, wherein the first determining means
further comprises means for determining a number of cell nuclei in
an image field having the optically-enhanced proliferation
substance therein, and the proliferation substance amount signal is
indicative of the number of optically marked cell nuclei having the
optically-enhanced proliferation substance.
7. An apparatus for determining a proliferation index of a cell
sample as defined in claim 6, wherein the second determining means
further comprises means for determining a number of optically
marked cell nuclei in the image field, and the cell nuclei amount
signal is indicative of the optically marked cell nuclei in the
image field.
Description
This is related to an application for Dual Color Camera Microscope
and Methodology For Cell Staining And Analysis to James W. Bacus
filed on the date of filing of this application.
BACKGROUND OF THE INVENTION
The invention relates to a system for performing a assay of a cell
sample to provide an accurate quantitative analysis of a
characteristic of the cells which have been sampled. More
particularly, the invention is directed to a system which receives
images of stained cells and enhances the cell images prior to
further processing to determine the proliferation index of the
enhanced cell images.
One of the problems which faces pathologists in their clinical
practice is that of determining whether a cell sample taken from a
patient during a biopsy procedure or the like is benign or
malignant. Although a surgeon may have a good intuition about the
type of tissue mass which he has removed, nevertheless he must
confirm his preliminary diagnosis with a histological examination
of the cell sample removed from the patient. The histological
examination entails cell staining procedures which allow the
morphological features of the cells to be seen relatively easily in
a light microscope. A pathologist after having examined the stained
cell sample, makes a qualitative determination of the state of the
tissue or the patient from whom the sample was removed and reaches
a conclusion as to whether the patient is normal or has a
premalignant condition which might place him at risk of a
malignancy in the future or has cancer. While this diagnostic
method has provided some degree of predictability in the past, it
is somewhat lacking in scientific rigor since it is heavily reliant
on the subjective judgement of the pathologist.
Attempts have been made to automate the cellular examination
process. In U.S. Pat. No. 4,741,043 to Bacus for Method and
Apparatus for Image Analyses of Biological Specimens, an automated
method and a system for measuring the DNA of cells are disclosed
which employ differential staining of the DNA in cell nuclei with a
Feulgen Azure A stain and image processing. While the system
provides an accurate assay of the cellular DNA its predictive power
for cell replication, a key indicator of the presence of cancer,
could be improved.
It is well known that cells follow a replication cycle; for a
further discussion of the cycle reference may be made to Pages
330-336 of McGraw-Hill Encyclopedia of Science and Technology, 6th
Edition, 1987. Most somatic cells of an adult human replicate at a
relatively slow rate, only rapidly enough to replace cells shed by
the body and lost to normal cellular wear and tear. At any instant,
most of those somatic cells are in the GO- or resting phase of the
replication cycle. When they leave the resting phase they enter the
Gl or first gap phase but are not yet producing extra DNA. Upon
becoming committed to the S-phase, however, they do produce other
material such as proliferation substances e.g. cyclin and other
S-phase proteins. The cells in the synthesis or S-phase are
actively synthesizing DNA and produce double the amount of DNA
normally contained in the cell nuclei in preparation for mitosis or
division of the cell nuclei during cell replication. A normal human
somatic cell contains 23 chromosome pairs and is in the diploid
state. The diploid state is also referred to as the 2N state. At
the time of replication the number of chromosome pairs increases to
46, double the normal amount in antic of cell division. The
chromosome state immediately before replication is referred to as
the 4N state. The cells then enter the second gap phase or G2 phase
in which little or no DNA is synthesized. Following the G2 phase is
the mitosis or M-phase in which the cells themselves divide. If the
cells are actively proliferating they may reenter the G1 phase.
Although DNA analysis may be adequate for estimating the number or
proportion of proliferating cells in normal cells or tissue, it
should be appreciated that this is not the case with malignant
cells, the very ones for which it often is important to know the
extent of proliferation. This is because malignant cells often have
increased amounts of DNA, even in the G0 phase, due to increased
chromosome content, and often increased of chomosomes. Therefore,
it is impossible to conclude with certainty from a DNA analysis
that a particular cell, e.g. one having 1.5 times the normal DNA
content, is a malignant cell with additional chromosomes, or
chromosome parts, or is a normal cell which is halfway through the
S-phase having only replicated one-half the DNA necessary for cell
division. Thus it is clear that an analysis method independent of
DNA, utilizing other markers, such as variously produced proteins
associated with S-phase proliferation and the cell division
process, has many advantages
It should also be appreciated that quantitating on a cellular
proliferation index has previously been performed by counting the
numbers of cells in a cell sample carrying an indicator or stain
for a proliferation substance. For instance, a well known method of
determining the proliferation index is to stain the cells with an
immunofluorescent dye which binds to cyclin and manually count the
fluorescent and non-fluorescent stained cells to determine the
proportion of cells having proliferation substance.
Another method of determining the proliferation index of cells is
the grain counting method; for a further discussion of this grain
counting method reference may be made to Pages 107-112 of The
American Journal of Pathology, Volume 134, No. 1, January, 1989. In
that method, tritiated thymidine is added to a cell culture growth
medium. Proliferating cells take up the tritiated thymidine and
incorporate it into DNA being synthesized in the cells. The cells
are then fixed and placed in proximity with a photographic
emulsion. Decay products of the tritium expose portions of the
emulsion. The exposed portions may be visualized as grains by
photographic development processes. Cells with overlying grains and
with non-overlapping grains, are then counted to determine the
proliferation index. One of the drawbacks of this method lies in
the fact that it is very time consuming. It is necessary that the
cells be harvested alive and kept alive long enough to take up the
tritiated thymidine. The cells must then be fixed and held in
proximity with the emulsion in order to expose it. Since relatively
low intensities of radiation may emanate from the cells, it may
take days or even weeks to obtain a latent image on the emulsion,
which must then be developed. In the meantime, the patient's
disease may be progressing.
One of the drawbacks of the prior art methods is that they are
prone to human error due to the tedium of counting the cells on a
microscope slide under high magnification. Often the people
examining the slides only are able to estimate the relative number
of cells which show a positive result for proliferation
substance.
The prior imaging systems have also suffered from the problem that
while they usually accurately identify the images of cell objects
in an image being processed they do not always accurately identify
boundaries of the cell objects being evaluated. This may be a
problem when an assay is being performed on the cell objects on the
basis of their image areas.
The prior art methods of quantitatively analyzing the cell samples
for proliferation substances could not be automated simply. This is
because it is necessary to determine a baseline value for the total
number of cells examined as opposed to the number of cells which
have proliferation substance. In order to make this type of
evaluation an automatic system must be able to recognize what
constitutes a cell or a cell nucleus. In order to solve this
baseline recognition problem the instant invention employs separate
stains for the cell nuclei and the proliferation substances. In
addition, the stains are separated spectrally so that they can be
readily distinguished by optical filters which are compatible with
them. The optical separation of the two components to be measured
makes the subsequent analysis of the cell images more convenient to
automate.
A similar difficulty is encountered in an image analysis based on
cell object areas when cell objects images overlap, touch or
otherwise share contiguous areas. In that case, what is actually a
double or triple object image may not be tallied properly resulting
in an inaccurate result or conclusion.
SUMMARY OF THE INVENTION
The present invention provides a rapid and convenient method and an
apparatus for practicing the method for determining the amount of a
proliferation substance in a cell sample. The cell sample may be a
tissue sample or a cell preparation. Tissue samples are frozen
sections or paraffin sections of connected cells. The cell
preparations are made from body fluids such as cerebrospinal fluid,
blood, pleural effusions and the like. Cell preparations may also
be made from needle aspirates of tumors, cysts or lymph nodes. Cell
preparations may also be made from touch preparations which are
made by touching a freshly microtomed surface of a piece of tissue
to a microscope slide to which the cells cling. In particular, the
apparatus and method employ a mouse PAP based staining system with
a rabbit anti-mouse bridging antibody, wherein mouse antibodies for
a proliferation substance such as cyclin or the antigen for Ki-67-.
are used. The PAP antibodies are complexed with an enzyme, in this
embodiment horseradish peroxidase (HRP). The cells are contacted
with the mouse primary antibody which binds only to portions of the
cells which have epitopes identifying them as proliferation
substance. After applying the bridging antibody, and the PAP
antibodies, a stain, in this embodiment 3, 3' diaminobenzidine
tetrahydrochloride (DAB), and hydrogen peroxide H.sub.2 O.sub.2 are
placed in contact with the cells having the antibody-HRP conjugate
bound to their proliferation substance sites. The HRP catalyzes a
chromogen forming reaction only at the areas where it is bound. The
catalyzed chromogen forming reaction produces a red-brown chromogen
precipitate bound to proliferation sites.
The cells are then stained with a counterstain, in this instance
ethyl green, which also is commonly known as methyl green. The
image of the cells is magnified in a light microscope and split
into a pair of separated images. The separated images are enhanced
by a pair of narrow bandpass optical filters. One of the narrow
bandpass optical filters preferentially transmits light having a
wavelength at the transmission peak of the counterstain thereby
producing an optically enhanced proliferation substance image which
only has background and the red-brown chromogen. The background of
the proliferation substance image is composed of the cell nuclei,
cytoplasm and the like which have substantially zero optical
density. The proliferation substance sites have a relatively high
optical density. Thus the only features which are easily
perceivable are the proliferation substance sites.
The other narrow bandpass optical filter preferentially transmits
in the regions of spectral absorption for both the red-brown stain
and the counterstain. This filter produces an optically enhanced
cell nuclei image of all nuclei, with and without proliferation
antigen.
The inventive apparatus senses the enhanced proliferation substance
image with a first monochrome television camera. The enhanced cell
nuclei image is sensed by a second monochrome television camera.
Analog signals representative of the images are fed to respective
image processors. The image processors convert the analog signals
to digitized arrays of pixels which are stored in internal frame
buffers.
When a tissue section is being examined the apparatus computes an
area of the proliferation substance image which has high optical
density, yielding an area measure for the proliferation substance
in that image field. When a cell preparation is being examined the
apparatus computes the proliferation index on the basis of the
percentage of cell nuclei having more than a threshold amount of
proliferation substance therein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an isometric view of an apparatus for determining a
proliferation index of a cell sample embodying the present
invention;
FIG. 2 is a block diagram of the apparatus of FIG. 1;
FIG. 3 is an elevational view of an optical conversion module of
the apparatus of FIG. 1;
FIG. 4 is a magnified view of a stained cell sample as seen through
the microscope of FIG. 1 without optical filtering;
FIG. 5 is a magnified view of the stained cell sample of FIG. 4 as
seen through a 620 nanometer narrow band optical filter which
yields a cell nuclei image;
FIG. 6 is a magnified view of the stained cell sample of FIG. 4 as
seen through a 500 nanometer narrow band optical filter which
yields a proliferation substance image;
FIG. 7 is a graph of the spectral response of a chromogen, a
counterstain and the narrow band optical filters;
FIG. 8 is a flow chart of a sequence of steps performed by the
apparatus of FIG. 1 in selecting a cell sample analysis mode;
FIG. 9 is a flow chart of a sequence of steps performed by the
apparatus of FIG. 1 in determining the proliferation index of a
tissue section cell sample;
FIG. 10 is a flow chart of the steps carried by the apparatus in
determining the proliferation index of a cell preparation cell
sample;
FIG. 11 is a screen display of the tissue screen;
FIG. 12 is a screen display of the cell preparation screen.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring now to the drawings and especially to FIG. -, an
apparatus embodying the present invention and generally identified
by numeral 10 is shown therein. The apparatus 10 comprises an
optical microscope 12, which may be of any conventional type but in
this embodiment is a Reichart Diastar or Microstar. An optical
conversion module 14 is mounted on the microscope 12 to enhance
optically a magnified image of a cell sample viewed with the
microscope 12. The optical conversion module 14, as may best be
seen in FIG. 3, has a cell nuclei sensing means comprising a cell
nuclei image optical enhancement unit 16. The cell nuclei image
optical enhancement unit 16 has a 620.+-.20 nanometer red narrow
bandpass optical transmission filter 18 and a television camera 20
for receiving a filtered image from the filter 18. A proliferation
substance sensing means comprising a proliferation substance
optical enhancement module 22 has a green 500.+-.20 nanometer
narrow bandpass optical transmission filter 24 and a television
camera 26 and is also part of the optical conversion module 14.
Each of the television cameras 20 and 26 generates a standard NTSC
compatible signal representative, respectively, of an enhanced cell
nuclei image and an enhanced proliferation substance image. An
image processing system 28 is connected to the television cameras
20 and 26 to receive the enhanced cell nuclei image signal and the
enhanced proliferation substance image signal and to store a cell
nuclei pixel array and a proliferation substance pixel array
therein. The image processor 28 is connected to a computer 32, in
the present embodiment, an IBM personal computer model AT for
processing of the cell nuclei and proliferation substance pixel
arrays.
The computer 32 includes a system bus 34, connected to the image
processor unit 28. An 80286 microprocessor 36 is connected to the
system bus 34. A random access memory 38 and a read only memory 40
are also connected to the system bus 34 for storage of information.
A disk controller 40 is connected by a local bus 44 to a Winchester
disk drive 46 and to a floppy disk drive 48 for secondary
information storage. A video conversion board 50 in this
embodiment, an EGA board having 256K bytes of memory, is connected
to the system bus 34 to control an instruction monitor 52 connected
to the EGA board 50. A keyboard processor 54 is connected to the
system bus 34 to interpret signals from a keyboard 56 which is
connected to the keyboard processor 54. A printer 58 is connected
to the system bus 54 for communication therewith. An X Y or image
field board 60 is connected to the system bus 34. The X Y board 60
also is connected to a slide holder of the microscope 12 to sense
the relative position of a slide 62 with respect to a microscope
objective 64 and thus identify a field being viewed. Included is a
Y position sensor 66 and an X position sensor 68. The Y position
sensor 66 is connected via a communication path 70 to the X Y board
60. The X position sensor 68 is connected via a communication path
72 to the X Y board 60. The microscope 12 also includes an eyepiece
76 in optical alignment with the objective 74 for magnification of
light forming an image of a cell sample on the slide 62.
The method of the instant invention is practiced by collecting a
cell sample, which may be in the form of a tissue section made from
a frozen section or a paraffinized section and having both cell
nuclei, cell fragments and whole cells therein. Alternatively, the
cell sample may be a cell preparation of the type which might be
taken from blood, pleural effusions, cerebrospinal fluid, or by
aspirating the contents of a cyst or a tumor. The cells of the cell
sample are placed on the slide 62 and fixed thereon. A monoclonal
antibody for a proliferation substance to be detected in the cells
is then placed in contact with them. The monoclonal antibody may
for instance be Ki-67 or may be an antibody for
5-bromodeoxyuridine, for cyclin or for other proteins which
indicate that cellular replication is occurring. The monoclonal
antibody selectively binds to all points on and within the cells
where the proliferation substance is present. The monoclonal
antibody also has bound thereto a bridging antibody and a
peroxidase anti-peroxidase complex. The anti-peroxidase comprises
an antibody which specifically binds to the enzyme peroxidase. The
peroxidase enzyme is bound to the antibody and held through the
chain of antibodies to the proliferation substance in the
cells.
In order to view the sites, a quantity of a mixture containing
hydrogen peroxide and 3, 3' diaminobenzidine tetrahydrochloride
(DAB) is applied to the cell sample on the slide. The hydrogen
peroxide and the DAB react to form a chromogen consisting of a
reddish-brown precipitate. The usual rate of reaction however is
relatively low. The peroxidase catalyzes the chromogen-forming
reaction only at the points where the peroxidase is localized.
Thus, chromogen is precipitated only at the points in the cells
where proliferation substance is present and the cells are
preferentially stained only at the points where they have
proliferation substance. After a period of about 15 minutes, the
unreacted DAB and hydrogen peroxide are removed from the cell
sample. The cells are then counterstained with methyl green (more
properly known as ethyl green) which preferentially binds with the
cell nuclei. Thus, cell nuclei are stained and the points within
the cell nuclei having proliferation substance are stained
reddish-brown.
The microscope slide 62 is then placed on a carrying stage of the
microscope 12 and the objective 64 is focused thereon. Light from
the objective 64 travels through the eyepiece 12 where at may be
viewed by an observer. In addition, the optical converter module 14
includes a beam-splitting mirror 80 which carries off approximately
90% of the light to other portions of the converter. The light is
fed to a dual prism dichroic mirror 82 which reflects a portion of
the light to the red filter 18. The remaining portion of the light
is filtered by the dichroic mirror 82 and fed to the green filter
24. The dichroic mirror 82 selectively passes light having
wavelengths greater than 500 nanometers to the filter 18 20. and
having a wavelength of less than 500 nanometers to the filter 24.
Thus, the dichroic mirror 82 acts as a first color filter before
the light reaches the color filters 18 and 24.
When the light passes through the filter 18, the filter 18
preferentially blocks light from the green stained cell nuclei and
provides a high contrast cell nuclei image to the camera 20. The
optical characteristics of the methyl green and the DAB as well as
the optical filters 18 and 24 are shown in the graph of FIG. 7. The
camera 20 then generates an NTSC cell nuclei image signal which is
fed to the image processor module 28. The image processor module 28
has an image processor 90 and an image processor 92. Each of the
image processors 90 and 92 is a model AT428 from the Datacube
Corporation. Similarly, the green filter 24, filter, provides a
high contrast proliferation substance image to the camera 26. The
camera 6 then feeds the proliferation substance image signal to the
image processor 92. Both of the image processors 90 and 92 contain
analog to digital converters for converting the analog NTSC signals
to digitized arrays of pixels which are then stored within internal
frame buffers. The internal frame buffers may be accessed via the
system bus 34 under the control of the microprocessor 36.
The image of the cell sample viewed through the eyepiece 12 is of
the type shown in FIG. 4 having a green cell nucleus 100, a green
cell nucleus 102, a reddish-brown cell nucleus 104 having
proliferation substance therein, a reddish-brown cell nucleus 106,
and a reddish-brown and green cell nucleus 108. As may best be seen
in FIG. 5, the cell nuclei are shown therein as they would appear
through the red filter 18, which causes all of the green cell
nuclei and the reddish-brown cell nuclei to darken and appear
prominently. As may best be seen in FIG. 6, the proliferation
substance image of the cell nuclei of FIG. 4 is shown therein with
the cell nuclei 100 and 102 being rendered substantially
transparent or invisible by the effect of the green filter 24. The
500 nanometer filter 24 transmits at an optical absorbing region of
the DAB and transmits at an essentially one hundred percent optical
transmission region of the methyl green. The 620 nanometer filter
transmits at an optical absorbing region of the DAB and at an
optical region of the methyl green. The cell nuclei 104, 106 and
108 having the reddish-brown chromogen deposited therein, which is
an indicator for the proliferation substance, appear clearly in
high contrast.
The image of FIG. 5 is stored in the internal frame buffer of the
image processor 90. The image of FIG. 6 is formed and stored in the
internal frame buffer of the image processor 92. It may be
appreciated that the pixel values for the images may be sliced
using standard image processing techniques to increase the contrast
between the cell nuclei and the backgrounds. That is, the areas of
high optical density in FIG. 6 such as the cell nuclei 104, 6 and
108 may be shown as being very dense and stored as high optical
density pixels, while the background areas 110 may be stored as
substantially zero optical density pixels in order to provide a
clear threshold or difference between the two areas. This is
particularly helpful when performing computations to determine the
proliferation index, since the system can differentiate more easily
between background and nuclei to be measured.
Once the images have thus been acquired by the system, the user, as
may best be seen in FIG. 8, is interrogated as to whether the
images are from a tissue section or a cell preparation. More
particularly, in a starting step 120, the system 10 next displays
an initial display screen 122 on the instruction monitor 52 and
thereby interrogates the user in a step 124 as to whether a tissue
section forms the basis for the image being processed. If the user
provides a positive response to the system, control is transferred
to a step 126 wherein a tissue section screen is displayed on the
instruction monitor 52. If the response is negative, control is
transferred to a step 128 where the user is questioned as to
whether the cell sample is from a cell preparation. If the response
is positive, control is transferred to a step 130 wherein a cell
preparation processing and result screen of the type shown in FIG.
12 is displayed on the instruction monitor 52. In the event that
neither of the selections is made, a step 132 is executed
transferring control to a HELP screen 134.
Referring now back to the step 126, it may be appreciated that the
screen of FIG. 11 is displayed in the step 126. The screen provides
a menu of functions at the right-hand side which are of the type
well known to users of automated cell analysis equipment. In
particular, the user may select a nuclear threshold function
wherein the user may specify the optical density or pixel value at
which the system determines for purposes of computation that a
particular pixel value is indicative of the presence of a portion
of a cell nucleus at that point. Furthermore, an antibody threshold
may similarly be set wherein the optical density of the image of
FIG. 6 is measured and a threshold is set indicative of the
presence or absence of antibody at a particular pixel address. In
addition, the user, once having set the thresholds, may then
instruct the system to display outlines or shaded areas, also known
as masks, of the cell nuclei and the antibodies in a display
nuc-anti masking function. Each of the masks is associated with the
particular cell nucleus by tag information stored in the system.
Once the masking step is finished, control is transferred to a
tissue section analysis step 140 which may be seen in more detail
in FIG. 9.
In FIG. 9, the 620 nanometer image of the type shown in FIG. 5 is
received by the camera in a step 150. The image is digitized in a
step 152 and a nuclear threshold value Tl for pixels indicating the
presence of the cell nuclei is selected in a step 154. Once the
threshold has been selected, pixels having a value less than the
threshold are reduced to zero leaving a high contrast pixel array
for further processing. The pixel array is transferred to the
computer system 32 where the number of pixels having values
exceeding the selected nuclear threshold value is counted to
provide a cell nuclei amount or count N1 in a step 156, which will
be used as a proliferation index denominator in later
processing.
Similarly, the image of the type shown in FIG. 6 is received by the
camera 26 in a step 160. The image is digitized by the image
processor 92 in a step 162. An antibody threshold T2 which has been
selected by the user reduces the background of the image to zero
and effectively isolates the pixels representative of antibody in a
step 164. The isolated pixels, that is those pixels having a value
greater than the preselected antibody threshold, are then counted
by the system 32 and a pixel count number N2 is provided in the
step 166.
Thus, it may be appreciated that steps 150 through 156 effectively
measure the area of the image field of FIG. 5 wherein cell nuclei
are found. The steps 160 through 166 effectively measure the area
of the proliferation substance in the image field of FIG. 6. The
system 32 in a step 168 then divides the area of the antibody by
the area of the cell nuclei and generates a quotient which is equal
to the proliferation index. The proliferation index is then
displayed on the tissue section screen as a percentage number. In
addition, the total nuclear area as computed in steps 150 through
156 is also displayed.
In the event that the user has indicated to the system in the step
128 that a cell preparation is being analyzed, control is
transferred to step 130 which may be seen in more detail, as shown
in FIG. 10. In a step 200, the cell nuclei image of FIG. 5 is
received by the camera 20. The cell nuclei image is digitized in a
step 202. The digitized image is then analyzed in a step 204 to
determine, using neighborhood labelling, what objects are to be
considered to be cell nuclei and what objects are not. The objects
to be considered to be cell nuclei are indicated by being
surrounded by boxes as displayed on the image monitor 30. In a step
206, if two or more of the objects are in contact with each other,
the operator is given the opportunity to have the system draw a
line of demarcation between them or to manually separate the images
himself. The labelling and separating are repeated in a step 208
until all cell nuclei are identified. In a step 210, a threshold
value Cl is then applied to the pixel arrays to isolate the pixels,
as was done in steps 154 and 164 previously.
Similarly, in a step 212, the proliferation substance image of FIG.
6 is received by the camera 26. The proliferation substance image
is digitized in a step 213 and the cell nuclei having more than a
threshold number of pixels are identified by neighborhood labelling
techniques as cell nuclei containing proliferation substance in a
step 214. If any cell nuclei images are touching they are separated
in step 215. Next, the remaining cell nuclei are also identified in
a step 216. The number of cell objects is counted in a step 217 to
yield a nuclear count N2. The images are combined in a step 218 and
displayed on the image monitor 30. The number of proliferation
substance nuclei N2 is then divided by the number of cell nuclei N1
in a step 220 to produce a proliferation index for the cell
preparation sample. The proliferation index is then displayed in
astep 222 on the cell preparation screen of FIG. 12.
It may thus be appreciated that the tissue section feature of FIG.
9 allows the proliferation index for a tissue section sample to be
easily and rapidly computed using stereological principles which
are standard in the field of microscopy. When tissue sections are
not used and stereological principles do not apply, the cells may
be counted by using the cell preparation technique.
Furthermore, the system provides considerable amplification for
determination of the proliferation index. The initial amplification
takes place when the proliferation substance is identified with the
chromogen and the cell nuclei are stained with the counterstain. A
second amplification takes place when the cell nuclei and
proliferation substance images are formed by filtering the light
through the filters 18 and 24. Further amplification takes place
when the threshold values for the antibody and the cell nuclei are
set providing high contrast images and high gain digital arrays for
further processing.
While there has been illustrated and described a particular
embodiment of the present invention, it will be appreciated that
numerous changes and modifications will occur to those skilled in
the art, and it is intended in the appended claims to cover all of
those changes and modifications which fall within the true spirit
and scope of the present invention.
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